Print version ISSN 1678-7757
J. Appl. Oral Sci. vol.19 no.2 Bauru Mar./Apr. 2011
Zubaidah Haji Abdul RahimI; Nalina ThurairajahII
IBSc Hons, PhD, Department of Oral Biology, Dental School, University of Malaya, Kuala Lumpur, Malaysia
IIBSc Hons, MSc, PhD, UCSI University, Dental School, University of Malaya, Kuala Lumpur, Malaysia
INTRODUCTION: Previous studies have shown that Piper betle L. leaves extract inhibits the adherence of Streptococcus mutans to glass surface, suggesting its potential role in controlling dental plaque development.
OBJECTIVES: In this study, the effect of the Piper betle L. extract towards S. mutans (with/without sucrose) using scanning electron microscopy (SEM) and on partially purifed cell-associated glucosyltransferase activity were determined.
MATERIAL AND METHODS: S. mutans were allowed to adhere to glass beads suspended in 6 different Brain Heart Infusion broths [without sucrose; with sucrose; without sucrose containing the extract (2 mg mL-1 and 4 mg mL-1); with sucrose containing the extract (2 mg mL-1 and 4 mg mL-1)]. Positive control was 0.12% chlorhexidine. The glass beads were later processed for SEM viewing. Cell surface area and appearance and, cell population of S. mutans adhering to the glass beads were determined upon viewing using the SEM. The glucosyltransferase activity (with/without extract) was also determined. One- and two-way ANOVA were used accordingly.
RESULTS: It was found that sucrose increased adherence and cell surface area of S. mutans (p<0.001). S. mutans adhering to 100 µm2 glass surfaces (with/without sucrose) exhibited reduced cell surface area, fuffy extracellular appearance and cell population in the presence of the Piper betle L. leaves extract. It was also found that the extract inhibited glucosyltransferase activity and its inhibition at 2.5 mg mL-1 corresponded to that of 0.12% chlorhexidine. At 4 mg mL-1 of the extract, the glucosyltransferase activity was undetectable and despite that, bacterial cells still demonstrated adherence capacity.
CONCLUSION: The SEM analysis confrmed the inhibitory effects of the Piper betle L. leaves extract towards cell adherence, cell growth and extracellular polysaccharide formation of S. mutans visually. In bacterial cell adherence, other factors besides glucosyltransferase are involved.
Key words: Bacterial adhesion. Glucosyltransferases. Streptococcus mutans. Scanning electron microscope.
Plants belonging to the genus Piper are reputed in the Indian Ayurvedic system of medicine for their medicinal properties9,31 and in folklore medicine of Latin America and West Indies. Piper betle L. (Piperaceae) leaves have a strong pungent aromatic flavour. In medicine, the leaves are useful in catarrhal and pulmonary infections31. The practice in India is to chew the leaves alone or with areca nut and other spices such as cardamom, clove and cinnamon, which act as ''breath fresheners'' and help in the prevention of halitosis. The phenolic constituent, allylpyrocatechol from the leaves showed activity against obligate oral anaerobes responsible for halitosis22. It has also been reported that the crude aqueous extract of Piper betle inhibits growth5 and adherence of early plaque settlers, which include S. mitis, S. sanguinis and Actinomyces sp. to saliva-coated glass surfaces23.
Other plant extracts like that of Syzygium aromaticum21, Polygonum cuspidatum27, Andrographis paniculata, Cassia alata, Chinese black tea, Guava, Harrisonia perforata10, Propolis Type 63 and cacao bean18 also have an antiadherence effect. The adhering property could contribute to the virulence activity of the bacteria11,12. The ability to adhere to oral surfaces so successfully is one of the factors that afford these bacteria a critical role in the formation of dental plaque which, eventually lead to oral diseases such as caries, gingivitis and periodontitis1,13,15. In previous studies, it has been reported that the crude aqueous extract of Piper betle L. leaves exhibit antivirulence and antibacterial activities towards S. mutans16. It was suggested that the antimicrobial activity is attributed to the hydroxychavicol component of the extract25.
Streptococcus mutans has been implicated as one of the primary causative agents of dental caries in human and experimental animals6,19. The bacteria after the initial colonization of the pellicle will attach strongly to the tooth surface by the extracellular polysaccharides (EPS). These extracellular polysaccharides are synthesized by the S. mutans in the presence of sucrose via the enzymatic action of one or more glucosyltransferases (GTFs). GTF activity may represent the virulence factor of mutans Streptococci7 and can be used as an index for determining the antivirulence activity of the extract. The presence of GTF-isoforms B, C and D as seen under electron microscopy within all layers of the in situ formed pellicle7 may affrm their signifcant role in plaque development. The results obtained from previous work17 have indicated the Piper betle L. extract affects the adhering capacity of S. mutans via inhibition of the GTF activity and hence the extracellular polysaccharide formation. This conclusion was based on biochemical and microbiological studies. The objectives of this study were to use scanning electron microscopy (SEM) to demonstrate visually the effect of the aqueous extract of Piper betle L. leaves in the presence and absence of sucrose on the cell adherence, cell growth and extracellular polysaccharide formation of S. mutans and to relate the effect of the extract on the GTF activity with bacterial adherence.
MATERIAL AND METHODS
Preparation of crude aqueous extract of the leaves of Piper betle
The leaves of Piper betle L. were obtained from one source in Mentakab, Pahang. Crude aqueous extract of the leaves was prepared according to Nalina and Rahim17 (2006). Before use the dried pellets were weighed, dissolved and diluted to the required concentrations using deionized distilled water. This is followed by fltration using 0.2 µm nylon syringe flters (Milipore, Billerica, MA, USA) which is for sterilization process26.
Preparation of bacterial suspension
Streptococcus mutans ATCC 25175 [American Type Culture Collection (ATCC), Manassas, VA 201808, USA] suspension was prepared according to Nalina and Rahim17 (2006). The bacterial stock which was kept frozen in glycerol at -70ºC before use was thawed at room temperature to revive the bacteria. The thawed stock was then dispersed in 30 mL Brain Heart Infusion (BHI) broth (Oxoid, Hampshire, England) before incubating it at 37ºC for 18-20 h. The number of bacterial cells in the suspension used in the study was standardized by adjusting the absorbance of the bacterial suspension spectrophotometrically (OD 550 nm) to 0.144. This absorbance is equivalent to 106 cells mL-1 23. To ensure that only pure culture of the stock are used in the study, the revived bacteria were consistently tested for purity by culturing them on BHI plates containing 5% blood.
SEM analysis on the adherence capacity and growth of S. mutans
SEM analysis was done to determine a) the effect of sucrose on the growth and adherence capacity of S. mutans to glass surface and b) the effect of Piper betle L. leaves extract on the adherence capacity and growth of S. mutans on the glass surface in the presence and absence of sucrose. The adherence capacity was determined from the bacterial cell population adhering to the surface of glass beads and the growth from the size and dividing appearance of the bacterial cells. The extracellular surface appearance of bacterial cells as viewed by SEM was also noted.
a) Effect of sucrose on the growth and adherence capacity of S. mutans to glass surface
The assay was based on a modifcation of the method developed by Ooshima, et al.18 (2000). 3 mL of Brain Heart Infusion (BHI) broth (Oxoid) containing 1% (w/v) sucrose (BDH) was pipetted into a vial containing sterile glass beads (diameter: 3 mm) (Merck, Darmstadt, Germany). This was followed by the addition of bacterial cell suspension containing S. mutans (equivalent to x106 cells mL1). The mixture was then incubated at 37ºC in an inclined position at an angle of 30 degrees for 18 h. This time duration allows the bacterial cells to be in the stationary phase. After the 18 h incubation, the glass beads were then washed with sterile deionized water and transferred into 1 mL of glutaraldehyde (Sigma Aldrich Co., St. Louis, MO, USA) (4%) to fx the cells and left overnight at 4ºC for the SEM analysis. SEM was used to view and record the cell population, cell growth and extracellular surface appearance of S. mutans. The assay was carried out in triplicate.
For blank control, the assay was repeated using BHI without bacterial inoculation. The blank control was to validate that the assay is devoid of bacterial contamination. For negative control, the experiment was repeated using BHI broth without sucrose.
b) Effect of Piper betle L. leaves extract on the adherence capacity and growth of S. mutans on the glass surface in the i) absence and ii) presence of 1% sucrose
The assay procedure in a) was repeated with the addition of the Piper betle extract at the respective concentrations (2 mg. mL-1 and 4 mg. mL-1) to the inoculated BHI broth. In the determination of i) the effect of plant extract in the absence of sucrose, the BHI broth devoid of sucrose was used and (ii) the effect of plant extract in the presence of sucrose, the BHI broth containing 1% sucrose was used.
Sterile deionized distilled water and 0.12% chlorhexidine were used in place of the plant extract for negative and positive controls respectively and the assays were also carried out using the BHI broth with or without sucrose. The beads from the experiments were also processed for the SEM study. All of the above assays were carried out in triplicates. There were 9 beads to be viewed under SEM for each experiment with the respective broths.
Preparation of the glass beads for SEM analysis
The glass beads that had been fxed overnight in glutaraldehyde were then processed for SEM analysis according to the following steps. First, the glass beads were washed with sodium cacodylate (Sigma Aldrich Co.) buffer (0.1 M at pH 7.4) and then fxed in 2% (w/v) osmium tetroxide (Merck) in the buffer solution overnight. The next day, the beads were gently washed in distilled water twice for 15 min and then dehydrated in an ascending series of ethanol (BDH Laboratories Supplies; VWR International, Lutterworth, Leicestershire, UK) concentrations (10, 20, 30, 40, 50, 60, 70, 80, 90 and 95%) for 15 min for each step. Following this, the beads were further dehydrated in 100% ethanol twice for 15 min and subsequently in ethanol:acetone mixture as follows, frstly, ethanol:acetone (3:1) for 15 min, then ethanol:acetone (1:1) for 15 min and followed by ethanol:acetone (BDH) (1:3) for 15 min. Finally, the beads were treated 3 times with pure acetone for 15 min.
The beads were then processed for critical point drying for about 2 h. Once the beads were completely dry, they were mounted onto metal stubs with double sided tape and then coated with gold. Three beads from each assay were then used for viewing using a scanning electron microscope (Phillips, Eindhoven, Netherlands).
Determination of the number of bacterial cells adhering to the glass surface
The number of cells adhering to the glass surface on a 100 µm2 area of the glass bead viewed at a magnifcation of 5,000x was calculated and the total number of cells was determined from an area of 1 mm2 of the glass surface. The area with high density of bacteria cells was used in the calculation of the number of cells (Figure 1). In total, 9 beads from 3 different assays per experiment were processed and analyzed.
Determination of bacteria cell surface area
The beads were viewed at a magnifcation of 10,000x. At this magnifcation, the size of the cell is magnifed and thus measurement is easily done. The size of the individual adhered bacterial cell was measured according its length (L) and width (W) in !m and expressed as the mean surface area [L±SD x W±SD] in !m2 of the total number of adhered cells from 9 beads used in the determinations.
Determination of the appearance of the extracellular surface
This was carried out by comparison with the appearance of the extracellular surface of S. mutans adhering to hydroxyapatite beads28.
Preparation of partially purified cellassociated GTF of S. mutans
Crude GTF was isolated from S. mutans ATCC 25175 strain in the research laboratory, Oral Biology Department, University of Malaya using the method described by Ooshima, et al.18 (2000) with slight modifcation17,21. The crude enzyme was then subjected to partial purifcation by ultra-fltration (Macrosep Omega membrane - mw cut off=300 kd; Pall Life Science, Exton, PA, USA). The crude enzyme was then centrifuged using the centrifugal flter (Macrosep Omega membrane; Pall Life Science) at 5,000 g for 20 min to remove residual cells and the supernatant collected was then referred as partially purifed crude enzyme. The partially purifed crude enzyme was kept in the freezer (-20ºC) to be used for further analyses.
Determination the effect of the extract towards partially purifed cell-associated GTF (CAG) activities
The reaction mixture used in the determination of the partially purifed cell-associated GTF activity was adapted from the method described by Mukasa, et al.14 (1979) and modifed to suit the experiment carried out in this study. The reaction mixture consists of 0.01% of thimerosal (Sigma Aldrich Co.), 1.4 g of sucrose, 34.3 mg of exogenous dextran (ICN Biomedicals, Aurora, OH, USA) and mixed in 100 mL of 5 mM sodium phosphate (Sigma Chemical Co.) buffer (pH 6.5). 100 mL of the partially purifed cell-associated GTF was pipetted into the reaction mixture to give a fnal volume of 2 mL. The mixture was then incubated at 37ºC. After 18 h (to allow the enzyme to catalyze the formation of glucan suffcient for quantifcation of glucose), the reaction was terminated by placing the tube in a boiling water bath for 5 min. After 5 min, the mixture w as centrifuged (Sigma 302K, Osterode, Germany) at 2,000 g for 10 min. The pellet obtained after centrifugation was collected and used in the determination of water-insoluble (WIS) extracellular polysaccharide content while the supernatant was discarded.
The pellet was washed 3 times each with 5 mL of distilled water and then uniformly suspended in 1 mL of distilled water by agitation. 50 mL of the WIS extracellular polysaccharide suspension was then pipetted into a clean test tube and assayed for glucose content.
The experiment was repeated by adding the plant extract at the respective concentrations (0-4.0 mg mL-1) to the enzyme reaction mixtures. Comparison was made between the activities of partially purifed CAG treated with plant extract and that without the treatment. The activity of the enzyme in the presence of 0.12% chlorhexidine (Sigma Aldrich Co.) was also determined using the same procedure.
Determination of glucose content of the WIS glucan
The determination of glucose was carried out according to the method by Dubois, et al.4 (1956). To 50 mL of the WIS glucan suspension, 50 mL of deionized distilled water was added and this was followed by the addition of 50 mL of phenol (Sigma Aldrich Co.) (80% w/v). The mixture was vortexed and subsequently 2 mL of concentrated sulfuric acid (R&M Chemicals, Essex, UK) was added to the mixture. This mixture was then allowed to stand for 10 min at room temperature before its optical density at 490 nm was measured. For the standards, a range of volumes (0-100 mL) of the stock glucose (Sigma Aldrich Co.) solution (1 mg mL1) was prepared to give a range of fnal concentration from 0 to 1 mg mL-1. For the blank control, 100 mL of deionized distilled water was used instead. The determinations were carried out in triplicates.
The glucose content determined corresponds to the amount of extracellular polysaccharide formed by the partially purifed GTF within the specifc time which was then used in the calculation of the GTF activity. The enzyme activity is expressed as µmol glucose in the extracellular polysaccharide produced per min (µmole glucose min-1)21.
Normality tests for all the data obtained were carried out using the Shapiro-Wilks analysis. The data for the comparison of the cell population and cell growth in the respective experiments were analyzed using a two-way ANOVA. For the comparison of the partially purifed CAG activities between the different experiments, a one-way ANOVA was used.
Effect of sucrose on the adherence capacity and growth of S. mutans on glass surface
Figure 2 shows SEM micrographs (10,000x magnification) for blank control (BHI without bacterial inoculation), negative control (inoculated BHI without sucrose) and test (inoculated BHI with sucrose) based on SEM viewing at. These images provide qualitative observations of the cell population which was used to determine the adherence capacity of S. mutans to glass surface. It was shown that there were no cells adhering to the glass surface in blank control (BHI without bacterial inoculation) (Figure 2A). This indicates that the experimental procedures are assured of the exclusion of bacterial contamination. The number of cells adhering to the glass surface when grown in the absence of sucrose was less (Figure 2B) compared to that in the presence of 1% sucrose as shown in Figure 2C. It was observed more cells were adhering to the glass surface and they are in aggregates. The extracellular surface of the cells appeared to have a layer of fuffy appearance spreading across the surface of the glass bead (Figure 2C).
The quantitative measurement of the bacterial cell population was carried out during the SEM viewing at 5,000x magnifcation. It was found that the number of cells adhering to the glass surface increases in the presence of sucrose and the increase was 6-fold higher than that in the absence (p<0.0001) (Table 1).
The cell surface area of the bacterial cells which was calculated based on the SEM viewing at 10,000x magnifcation is shown in Table 2. It was found that there was an increase (33%) in the cell surface area of the bacterial cells when grown in the presence of sucrose compared to that grown in the absence (p<0.0001).
Effect of Piper betle L. leaves on the adherence capacity and growth of S. mutans in the presence and absence of 1 % sucrose
The SEM micrographs in Figure 3 show the effect of the different concentrations of the Piper betle L. leaves extract on bacterial cells grown in the BHI broths without sucrose and with 1% sucrose based on the analysis at 10,000x magnifcation (without sucrose in the presence of 2 mg mL-1 extract -Figure 3A; with 1% sucrose in the presence of 2 mg mL-1 extract - Figure 3B; without sucrose in the presence of 4 mg mL-1 extract - Figure 3C; and with 1% sucrose in the presence of 4 mg mL-1 extract - Figure 3D). The effect of the extract appeared to be concentration dependent where the number of cells adhering to the glass surface decreases with the increase in the concentration of the extract. It was shown that sucrose also has an effect on the growth and adherence of the bacterial cells.
Effect of the Piper betle L. leaves extract on the adherence capacity of the S. mutans
It was found that the addition of the Piper betle extract to the BHI broths reduces the cell population of S. mutans adhering to the glass surface. The reduction was observed both in the absence and presence of sucrose. The number of cells adhering to the glass surface reduced by 27% (p<0.0001) when grown without sucrose and in the presence of 2 mg mL-1 of the extract compared to that in the absence of the extract. A similar reduction in the number of cell adhering to the glass surface was observed for those grown in the presence of sucrose. Increasing the extract concentration to 4 mg mL-1 resulted in a further decrease of the cell population, with a reduction of more than 80% both in the absence and presence of sucrose (Table 1). No bacterial cells were observed to be adhering to the glass surface when chlorhexidine was used instead of the extract.
Effect of the Piper betle L. leaves extract on the growth of S. mutans with and without sucrose
The cell surface area of the bacterial cells grown in the presence of the extract, with and without sucrose based on the SEM viewing at 10,000x magnifcation is shown in Table 2. It was found that the bacterial cells grown in the presence of 2 mg mL-1 of the extract without sucrose exhibited cell surface area that is not signifcantly different from those grown in the absence of the extract. Increasing the concentration of the extract to 4 mg mL-1 decreases the cell surface area by 30% (p<0.0001).
It is shown in Table 2 that the cell surface area of the bacterial cells grown in the presence of 1% sucrose and 2 mg mL-1 of the extract is reduced by 12% compared to that of those grown without the extract (p<0.0078). The bacterial cells also appeared to aggregate and exhibit fuffy extracellular surface but to a lesser extent compared to those grown in the absence of the extract (Figure 3B versus Figure 2C). There was an almost 50% reduction in the cell surface area when the bacterial cells were grown in the presence of a higher extract concentration (4 mg mL-1) (p<0.001).
It was observed that the effect of the different concentrations of the extract is not greatly infuenced by the presence of sucrose. The cell surface area of the bacteria in the presence of 1% sucrose and 2 mg mL-1 extract only increased by 12% compared to the cell surface area of those in the absence of sucrose (p<0.0002). At 4 mg mL-1 concentration of the extract, the bacterial cells attained almost similar size both in the presence and absence of sucrose.
Effect of the Piper betle L. leaves extract on the activity of partially purifed cellassociated GTF (CAG)
Table 3 shows the effect of the extract on the partially purifed CAG activity. The activity was expressed as µmol glucose content of the extracellular polysaccharide per minute and was found to decrease in the presence of the extract. The inhibitory effect was found to be concentration dependent and was more signifcant at higher concentrations of the extract. At 0.5 mg mL-1 of the extract, the partially purifed CAG activity was reduced by 29% (p<0.0001) when compared with the negative control (without the plant extract). At a higher concentration of the extract (1 mg mL-1), the activity was reduced by 35% (p<0.0001) and at 2.5 mg mL-1 of the extract, the decrease was 70% (p<0.0001), the latter being almost similar to that observed with chlorhexidine (p>0.05). Increasing the concentration of the extract to 4 mg mL-1 resulted in undetectable partially purifed CAG activity.
In this study, the SEM was used to determine both visually and quantitatively i) the effect of sucrose on growth and adhering property of S. mutans and ii) the effect of Piper betle extract on S. mutans in the presence and absence of sucrose. The SEM study can also be used to show the appearance of the extracellular surface of the cells in the presence of the extract with or without sucrose. Previous studies2,8,24,30 had employed spectrophotometric technique that allows for quantitative determination of cell population (CFU mL-1) to relate bacterial population and growth.
The increase in cell population adhering to glass surface in the presence of sucrose compared with those in the absence indicates that sucrose has a role in the adhering property30 and growth of S. mutans2,8,24,30,32. S. mutans is a secondary colonizer of dental plaque featuring cariogenic properties including cell adhesion, cell surface hydrophobicity and glucan synthesis activities21. The initial adherence or attachment of bacteria to the tooth surfaces may involve hydrophobic and ionic interactions between the receptors on the host surface and the adhesins on the cell surface of the bacteria11,12. The extracellular polysaccharide (glucan) which is formed favorably in the presence sucrose may be responsible in enhancing the adherence of more bacterial cells to the host surface.
The size of the bacterial cells is refected by the cell surface area. It was found to be increased by 33% in the presence of sucrose. Sucrose is a good substrate for GTF as its hydrolysis produces energy suffcient for the polymerization of glucose in the formation of glucan, an extracellular polysaccharide. Thus its presence in the BHI broth helps to promote the activity of this enzyme. This explains for the formation of the extracellular polysaccharide which gives fuffy appearance of the extracellular surface of S. mutans when grown in the presence of sucrose.
For all the experiments to show the effect of the extract of Piper betle L. leaves towards S. mutans, sub-minimal inhibitory concentrations (sub-MICs) (2 mg mL-1 and 4 mg mL-1) were used. These concentrations were used to ensure that the observed reduced number of adhering cells was not attributed to cell death. This parameter has been used as a reference for bacteriostatic effect of plant extract20.
It was shown that the Piper betle L. leaves extract reduces adherence of the bacterial cells to the glass surface, and this is further confrmed by the calculated cell population visualized by SEM. It was thought that the reduced adherence is attributed to extracellular polysaccharide not being formed effciently, which is due to reduced GTF activity in the presence of the extract.
In this study, it was shown that there are bacteria though few in number adhering to the glass surface despite the undetectable GTF activity in the presence of 4 mg mL-1 of the extract. In the presence of 0.12% chlorhexidine there were no bacterial cells adhering to the glass surface and at that concentration, the GTF activity corresponds to that of 2.5 mg mL-1 of the extract and, cell adherence was observed in the presence of the later. This suggests that GTF is not involved and that other factors such as hydrophobic and ionic interactions maybe involved in the adherence or attachment of bacteria to the tooth surfaces which, is in agreement with what had been reported in previous work11,12. However, it has been shown that the cell surface hydrophobicity of S. mutans is reduced in the presence of the plant extract16. The crude Piper betle L. leaves extract may have infuenced the adhesion between the cell surface of the bacteria and the host surface via ionic interaction which could be responsible for the adherence effect demonstrated at 4 mg mL-1 extract concentration.
It was also shown that the Piper betle L. leaves extract has an infuence on the cell surface area (size) of the bacterial cells. The presence of sucrose appears not to have effect on the cell surface area of bacterial cells in the presence of the 4 mg mL-1 extract as with or without sucrose, the bacterial cells appear to attain similar cell surface area. This indicates that bacteria grown in the presence of the extract could experience environmental stress, which could infuence its ability to use nutrients effciently and thereby slows down its growth5,29. It has been reported that the aqueous Piper betle L. extract exhibited similar effect towards other oral bacteria for example, S. sanguinis, S. mitis and Actinomyces sp.5.
The SEM analysis provided visual (qualitative) and quantitative evidence that the Piper betle L. leaves extract has a reducing effect on cell adhesion, cell growth and extracellular appearance of S. mutans. The effect of the Piper betle L. leaves extract on the GTF activity and adherence capacity of S. mutans suggests the involvement of other factors in the adherence of bacterial cells to the glass surface.
This project was supported by research grants (Vote F and IRPA) provided by the Malaysian government. The authors would like to acknowledge Ms Zubaidah Abu Hassan of the Electron Microscopy Unit, Medicine School, University of Malaya for their assistance in the electron microscopy procedure.
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Zubaidah Hj Abd Rahim (PhD)
Department of Oral Biology, Faculty of Dentistry - University of Malaya
50603 - Kuala Lumpur - Malaysia
Phone: 603-79674851 - Fax: 603-79674536
e-mail address: firstname.lastname@example.org
Received: June 16, 2009
Modifcation: November 24, 2009
Accepted: March 19, 2010